This study aimed to: i optimize prechilling conditions by prechilling seeds at a range of moisture contents 28-34 % for different durations X to X+6, where X is the duration necessary to
Trang 1Original article
Claudine Muller Elyane Laroppe a Marc Bonnet-Masimbert
a Inra Centre de Nancy 54280 Champenoux, France
b
Inra Centre d’Orléans 45160 Ardon, France
(Received 11 February 1998; accepted July 1998)
Abstract - Techniques for breaking dormancy of beechnuts (Fagus sylvatica) have been greatly improved over the last 20 years A
major improvement has been the introduction of a new procedure involving prechilling after rehydration of the seeds to a precise mois-ture content (m.c.) (28-30 %) This allows long-term storage of non-dormant beechnuts This study aimed to: i) optimize prechilling
conditions by prechilling seeds at a range of moisture contents (28-34 %) for different durations (X to X+6, where X is the duration necessary to obtain the germination of 10 % of the viable seeds under stratification in a wet medium at 3 °C); ii) follow changes in
the germination ability of prechilled seeds during 3 years of storage The germination capacity of beechnuts was tested just after
prechilling, after prechilling and drying down to 8 %, and after prechilling, then drying and storage The best maintenance of the ger-mination percentage during storage was obtained when seeds were prechilled at 30 % m.c for X+2 weeks A damaging effect of dry-ing was observed when the moisture content of the seeds during prechilling was high (34 %) and the duration long (X+6) When
prechilling was applied after storage the best results were obtained at 32 % m.c (© Inra/Elsevier, Paris.)
Fagus sylvatica / beechnut / seed / dormancy / storage
Résumé - Progrès dans la conservation des faines (Fagus sylvatica L.) prétraitées : influence de la teneur en eau des faines et
de la durée de prétraitement Les techniques de levée de dormance des faines (Fcagus sylvatica) ont été grandement améliorées au cours des vingt dernières années Un progrès majeur a résulté de l’introduction d’une technique de prétraitement après réhydratation
des semences à un niveau de teneur en eau (TE) contrơlé (28-30 %) De plus, ceci a ouvert la voie à la conservation à long terme des faines dans un état non-dormant Notre étude visait à : i) optimiser les conditions du prétraitement : TE des faines variant entre 28 et
34 % et durées variant entre X et X+6 (ó X représente le nombre de semaines permettant la germination de 10 % des faines viables,
en condition de stratification dans un milieu humide à 3 °C) ; ii) suivre pendant trois années de conservation l’évolution de la faculté
germinative des faines ainsi prétraitées Des tests de germination ont été effectués juste après le prétraitement, après prétraitement et
séchage à TE 8 %, et après prétraitement, séchage et conservation
Le meilleur maintien de la faculté germinative est obtenu à la suite d’un prétraitement à TE 30 % pendant X+2 semaines Le séchage
occasionne une baisse de faculté germinative lorsque le prétraitement a été effectué à TE 34 % et pour la durée la plus longue (X+6)
Lorsque le prétraitement est appliqué après conservation, les meilleurs résultats sont obtenus à TE 32 % (© Inra/Elsevier, Paris.)
Fagus sylvatica / faines / semence / dormance / conservation
*
Correspondence and reprints
cmuller@nancy.inra.fr
Trang 21 INTRODUCTION
The European beech (Fagus sylvatica L.) is an
impor-tant timber species often used in forestry Beechnuts, like
many other forest seeds, possess embryo dormancy which
responds to cold The dormancy can be particularly deep
because it may require 4 to 20 weeks of prechilling at a
temperature of 1 to 5 °C before it is broken [6, 12, 18, 23].
This dormancy can vary greatly from year to year, from
one seedlot to another and, even within a seedlot, from
one seed to another
Traditionally, beechnuts are prechilled at 3 °C in a
moist medium (cold stratification) but the seeds will also
germinate at this temperature as soon as dormancy is
bro-ken [ 16] This premature germination during chilling
cre-ates problems for sowing Therefore, Suszka and Zieta
[20] proposed a modification to the traditional treatment
by interrupting the pretreatment when 10 % of nuts have
germinated This duration, expressed in weeks, is
desig-nated as ’X’ It is clear that after X weeks of chilling,
dor-mancy is not completely released and so seedling
emer-gence is generally neither uniform nor complete.
Moreover, since germination is considered an irreversible
process, there is no way to dry the seed after prechilling
without serious damage to seeds in the early stages of
germination.
In 1975, to overcome the drawbacks of the classical
cold stratification with a medium, a new procedure of
prechilling, based on the control of seed moisture content
(m.c.) without a medium was developed for beechnuts in
Poland [17] and further elaborated in France [11, 12] In
this procedure seeds are moistened during prechilling to a
m.c of 28-30 % and then maintained at 3 °C for 2 weeks
longer (i.e X+2 weeks) than for classical stratification
This precise m.c is enough for dormancy breaking to be
achieved, but it does not allow seeds to germinate As a
consequence, seeds can be dried without damage after the
prechilling which can be applied immediately after
har-vest, or after a period of storage and before sowing [10].
Since 1976 we have conducted many experiments and
obtained very promising results for storing non-dormant
beechnuts for up to 8 years [11, 12, 14, 22, 23] In recent
years the new methodology has been applied in France on
a large scale by the ’Office National des Forêts’ and by
seed companies In 1990 one of the largest French seed
companies (Vilmorin, Angers) applied the procedure to
10 tons of beechnuts and achieved an average
germina-tion (among different seedlots) of 60-70 % These
non-dormant beechnuts were successfully used over the next
18 months
The purpose of this study was: i) to optimize
prechilling conditions, with respect to both seed moisture
content and the duration of chilling and ii) to follow
changes germination of prechilled seeds for 3 years
of storage in relation to these conditions
In addition, we tested whether the same dormancy breakage conditions would be equally effective before or
after storage, i.e whether seeds were stored in a
non-dor-mant or dormant state.
2 MATERIALS AND METHODS
2.1 Seed material
A Danish seedlot (45 kg), supplied by the Tree
Improvement Station (Humlebaek) was used for the
experiments Upon arrival, in December 1993, seed m.c.
was 21.6 % and viability 77 % The degree of dormancy
(X) was determined in stratification just after arrival
(before and after drying): 6 weeks were necessary to
obtain 10 % of germination at 3 °C for wet beechnuts and
only 4 weeks for dried beechnuts
2.2 Prechilling, drying and storage conditions
A part of the seedlot (25 kg) was prechilled just after arrival The following m.c and prechill durations were
applied in a factorial design: 28, 30, 32 and 34 % m.c and
X, X+2, X+4 and X+6 weeks for the durations (X = 6
weeks) The longest duration thus corresponds to 12 weeks of pretreatment In this paper ’prechilling’ will
designate prechilling without medium in contrast to
’stratification’ which designates a prechilling into a wet
medium After pretreatment this first lot was dried at
room temperature to 7-8 % m.c and stored at -7 °C The remainder (20 kg) of the seedlot was stored in a dormant
state for 18 months at 7-8 % m.c and -7 °C; they were
then prechilled at 30, 32 and 34 % m.c for X+2, X+4,
X+6 and X+11 weeks just before sowing.
2.3 Germination tests
Germination tests were performed on four replicates of
50 seeds (randomized block design), in darkness, in the
laboratory on moist filter paper at 3°/20 °C (16 h + 8 h).
These alternating temperatures are generally used in our
laboratory because they have shown a good correlation with germination in nursery conditions
The results are expressed as germination percentage
(GP) in the laboratory or seedling emergence percentage
(SEP) in the nursery and mean germination time (MGT)
Trang 3(or velocity) [9]
formula:
with n = number of germinated seeds after ti days and
N = total germinated seeds at the end of the test.
The seeds were tested in the laboratory, after receiving
the dormancy breaking treatments, before and after
dry-ing and after 1, 2 and 3 years of storage
After 18 months of storage, the germination and
nurs-ery emergence of seeds that had been prechilled before
storage was compared with those pretreated after storage
In the nursery only the best treatments (according to the
laboratory test after I year of storage) were sown, i.e 30
and 32 % m.c during X+2 and X+4 weeks for seeds
stored prechilled; 32 and 34% m.c during X+2 weeks for
seeds prechilled after storage
2.4 Statistical analyses
Statistical differences for the number of germinated
seeds per replicate and MGT were submitted to analysis
of variance according to a two-way classification with
interaction (procedure ANOVA, SAS) Bonferroni’s
mul-tiple range test at 0.05 level of probability was applied to
compare means of significant main factors or interaction
3 RESULTS
The results will be presented in three stages: 1) just
after prechilling, 2) after prechilling and drying and 3)
after prechilling, drying and storage for 18 months in
comparison to non-prechilled seeds also stored for 18
months
of prechilling
moisture content and duration
Two series of tests were performed immediately after the arrival of the seeds in the laboratory.
3.1.1 Results obtained just after prechilling
The results are given in table I Germination was sig-nificantly higher (average 80 %) at m.c varying between
30 and 34 % than at 28 % m.c where GP was 64.3 %
There was no significant difference between either
aver-age GP or MGT at 30, 32 and 34 % m.c For a short
dura-tion of prechill, such as X weeks, good germination was
already obtained at 30 and 32 % m.c which suggests that
breaking of dormancy starts earlier at 30 and 32 % m.c.
than at the other m.c (28 and 34 %) However, X weeks
(GP 61.2 % on average) was not sufficient to completely
break the dormancy No significant difference was
observed between X+2, X+4 and X+6 weeks (GP around
80 %) For the speed of germination, the best results were
obtained at 30-32 % m.c and with the duration X+4 weeks (individual treatment values not shown).
3.1.2 Results obtained after drying
The results are given in table II The GP before and after drying, averaged for the factors ’duration’ and
’moisture content’, are also summarized in figure 1
Immediately after drying, we generally observed a slight
but significant decrease of GP: 5 to 10 % on average,
depending on the treatment - except for X weeks where there was a systematic increase (compare tables I and II).
The decrease was greater at the longest prechill duration,
X+6 weeks and the m.c high (34 %) It is possible that
some seeds were in the early stages of germination and
Trang 4higher m.c., stronger negative
drying.
After drying (table II) there was no effect of prechill
duration in the range of X to X+4 weeks whatever the
moisture content The best values for GP and MGT were
always obtained in the range of 30-32 % (even 34 %) for
the m.c., and X, X+2 and X+4 for the duration: GP
var-ied between 74.5 and 79.5 % and MGT was around 12
days (individual treatment values not shown).
3.2 Storage for 18 months:
comparison of seeds prechilled before storage
and seeds prechilled after storage
3.2.1 Laboratory tests
With regard to the GP of seeds prechilled before
stor-age (table III), the best results were obtained at 30 and 32
lower at 28 % m.c whatever the duration At 34 % m.c.,
GP decreased by 48 % at the longest duration of
prechilling (X+6) For seeds pretreated after storage (table V), the highest Gps were obtained at a moisture
content slightly higher than for seeds prechilled before
storage i.e 32 and 34 % m.c The duration X+2 seems to
be sufficient but X+4 weeks and even longer periods can
be used without any significant decrease in germination
as opposed to what we observed for prechilling before
storage
In case of the MGT (tables IV, VI), the longer the
pre-treatment the faster the germination MGT was particu-larly low for seeds prechilled after storage, most probably
because they were sown moist (30-34 % m.c.) as
opposed to seeds prechilled before storage which were
sown at their m.c in storage, i.e 8 % m.c.
Trang 5Results for seedling emergence obtained in the nursery
(figure 2) were similar to those obtained in the laboratory
(tables III-VI) Even if there were no significant
differ-ences in SEP between the six treatments (applied before
or after storage), overall the behaviour of seeds stored after being prechilled seemed slightly better than that of
seeds classically prechilled just before sowing However,
the rate of seedling emergence was slightly faster with the seed prechilled after storage (MGT = 16.6) than with seed
prechilled before storage (MGT = 17.7), probably
because they were sown at a higher m.c.
3.3 The changes in germination
of prechilled seeds during storage
Figure 3 shows all the results obtained in the
laborato-ry during the 3 years of storage Good stability was
obtained at 30 and 32 % m.c with durations X and X+2 weeks with no significant loss of germination after 3
of storage However, the best maintenance of GP
Trang 6prechilling performed
for X+2 weeks When the duration was longer than X+2
weeks, there was a large decrease of GP after the 2nd year
of storage, which was significant at 32 and 34 % m.c At
28 % m.c., even though GP was never high, it did not
decline
Poor results were generally obtained for X+6 weeks of
prechilling and to some extent for X+4 weeks, especially
at 32 and 34 % m.c This decrease was already noticeable
after 1 year of storage.
4 DISCUSSION
Following the ideas of Suszka [ 17, 20], beechnuts have
now been successfullly stored prechilled for up to 30
months [11], 42 months [12, 14] and even 6 to 8 years [5,
10, 13].
Since the 1990s, the drying and storage of prechilled
seeds has also been successfully developed for seeds of
other hardwood species, particularly Fraxinus excelsior
[25] and Prunus avium [ 14] In the case of conifers,
stor-age of prechilled seeds has been difficult, particularly
when they were redried to a m.c of 10 % or less [ 1, 3, 4,
7, 27] In 1995, Jones [8] succeeded in redrying Picea
without any loss of viability
some dormancy was reinduced during storage Recently,
we have obtained positive results with prechilled Douglas
fir seeds stored at 6 % m.c (Muller, unpublished results);
neither re-imposition of dormancy nor loss of viability
were observed after 2 years of storage
In the case of beechnuts, the key is in prechilling at a
controlled moisture content which permits the breaking
of dormancy without allowing the germination The
pro-posed prechilling m.c have varied from one author to
another However, in all cases, the values range between
28 and 32 % [2, 5, 12, 13, 18, 20, 21, 23].
In the present study, our first objective was to optimize prechilling conditions before drying and storage Just
after prechilling (table I), the high GPs (around 80 %) were obtained at m.c between 30 and 34 % m.c At 28 %
m.c., GP was significantly lower (64.3 %), which
sug-gests that at this m.c not all the seeds have had their dor-mancy broken Therefore, the prechilling moisture
con-tent must not drop below 30 %
To take into account the heterogeneity of the seedlot,
the prechilling duration can be varied for different seed-lots For dormancy breaking without further storage, durations ranging from 4 to 20 weeks have been
Trang 7pro-posed Recently, [2] reported
cold prechilling of 16-20 weeks is required to obtain the
optimal dormancy breakage in freshly harvested
beech-nuts Gille and Nowag [5], advocated 14 weeks for all
seedlots Other authors [12, 18, 20], however, prefer to
adjust the duration of the prechilling to the estimated
degree of dormancy of each seedlot As already
men-tioned, this estimate refers to an X value (in weeks) [20]
where X is, for a given seedlot, the duration necessary to
obtain 10 % of germination of viable seeds under
condi-tions of stratification in a wet medium In our
experi-ments, increased germination was obtained between X+2
and X+6 weeks (table I) with no significant difference in
this range of durations if there was no further storage
When storage is planned, it is necessary to dry the
seeds We have shown here that this drying causes a
decrease (table II, figure 1) of the GP However, for the
shortest duration (X), which was clearly insufficient for
dormancy breakage,
after drying Similar stimulation of seed germination by reducing water content has also been observed in the
mature or immature seeds of many species (see review by
Thomsen [24]) It has also been observed in dormant tree seeds, Aesculus hippocastanum [15] and Fagus sylvatica
[17] According to Thomsen [24], drying can replace part
of the cold requirement for beechnuts In her case drying
the beechnuts to 8 % m.c gave the same results as 3 to 4 weeks of cold stratification
Our experiment confirms that drying after prechilling
has the same positive effect on the seeds whose
dorman-cy was incompletely eliminated after X weeks In our
case, such treatment replaced two weeks of cold and therefore no significant difference was observed between
X and X+2 weeks after drying (table II) as opposed to a
significant difference before drying (table I) On the other
hand, seeds with the longest prechilling (X+6 weeks)
Trang 8sig-nificantly drying, especially
bined with a high m.c (34 %) In the latter case, a loss of
about 40 % GP is observed According to Derkx and
Joustra [2], it is possible that early germinative events,
which start at the end of dormancy breaking, reduce the
tolerance to dessication
After prechilling, the beechnuts were dried at a
mod-erate temperature (around 18-20 °C) down to 7-8 % m.c.
Prechilled beechnuts seem to be more sensitive to drastic
drying (e.g drying to moisture content around 5 %) than
dormant beechnuts (Muller, unpublished results).
However this needs further investigation.
The current experiments demonstrated that beechnuts
can be successfully stored in sealed containers at -7 °C
for at least 3 years (figure 3) If they are to be stored in a
non-dormant state, the best maintenance of GP was
observed after a prechill at 30 % m.c for X+2 weeks,
which confirms previous results [5, 12, 13].
Another outcome of our experiments concerns the
comparison between conditions for dormancy breakage,
when the treatment is applied before or after storage In
fact, several possible combinations between prechilling
and storage have been proposed for beechnuts [10, 14]:
prechilling before storage, during storage [21] Until now,
the same prechill conditions have been proposed for these
different cases: 30-32 % m.c for X+2 weeks [19, 22, 23].
In the present experiment, prechilling, whether it was
applied before or after storage, led to relatively similar
results, in the laboratory (tables III-VI) and in the nursery
(figure 2), after 18 months of storage However, when
prechilling was applied after storage (table V and VI),
there was some advantage from increasing the moisture
content to 32-34 % m.c., i.e 2 % higher than when seeds
were prechilled before storage (table III and IV).
In conclusion, the ability to dry and store non-dormant
seeds has definitely opened new possibilities in the
han-dling and preparation of dormant species The integration
of dormancy release treatments with seed storage for
deep dormant hardwood seeds ensures the availability of
non-dormant seeds that are able to germinate without any
further pretreatment even after long storage It brings a
flexibility to a situation where the constraints (including
the necessary variability of the seeds from very diverse
trees) are numerous and represents an important advance
in the technology of forestry seeds
Acknowledgements: This work was supported by the
European Union through the project AIR2-CT93-1667:
’A multidisciplinary approach to the understanding and
efficient handling of seed dormancy in tree species’ The
authors are grateful to Simon Hawkins for critical reading
of the manuscript.
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